The purpose of the proposed work is to study by NMR active sites of proteins (antagonists, inhibitors, severins) that interact with receptor- like proteins (receptors, proteinases, cytoskeletal proteins), to mutate the residues in the active sites to change their structural and functional properties, to gain information for polypeptide design. The goal is to learn about principles of such interactions, to identify receptor binding sites, and to characterize the conformation of the receptor interfaces in the free form and the complex. In this context, changes of the mobility of the proteins upon binding will be investigated. It is anticipated that the knowledge about the conformation of the binding site in the free protein and in the complex will provide clues for designing small molecules that would have similar binding properties as the protein itself (agonists and antagonists). The research will be carried out with the following classes of proteins. (i) The structure of the proteinase inhibitor eglin c will be investigated by mutations, NMR experiments, structure calculations, studies of the internal mobility and computer simulations. The complex of eglin c with subtilisin Carlsberg will be used as a model for a protein receptor complex. The interface with the proteinase will be followed by direct recording of the 2D 1H-15N heteronuclear correlated spectrum at different conditions of relative concentrations and pH. Isotope labeled eglin is available. The binding properties can be modulated by variation of the pH so that conditions can be optimized for different types of experiments. (ii) The RGD proteins kistrin and decorsin are antagonists of platelet aggregation. They bind to the platelet receptor GPIIbIIIa. Refined structures of both proteins will be determined. The active site is relatively mobile in the free proteins. Efforts will be made to obtain information on the conformation in the receptor complex. A truncated extra cellular domain of the receptor (trGPIIbIIIa) will be available for studies of the interaction of the receptor with the disintegrins. (iii) The conformation of domain l of the actin binding protein villin will be determined by NMR. Constructs of domains 1+2 and 2+3 will also be investigated. The interaction of domain l with actin will also be investigated, as will be the binding of Ca2+ and of phospholipids to villin. To perform this research, advanced multidimensional multiple resonance experiments will be employed.